Sphingolipids are indispensable components of myelin, synaptosomes and other membranes in the brain. The unequivocal elucidation of metabolic pathways of these compounds are essential for our understanding of brain metabolism and function. The proposed project is designed to examine the role of 3-ketoceramides, 3-ketocerebrosides and 3- ketospingomyelin in the biosynthesis of corresponding sphingolipids in the brain. The sphingolipids labelled with (C14-1) lignoceric acid, (C14-1) cerebronic acid, or (C14-1) stearic acid moiety will be synthesized and converted to the corresponding 3-keto derivative. The latter reaction will be carried out quantitatiely with 2,3-dichloro-5,6- dicyanobenzoquinone. These (C14) labelled 3-ketosphingolipids may be further converted to doubly labelled 3-ketodihydrosphingolipids. This will be accomplished by the introduction of H3 in 4,5 position of dihydrosphingosine moiety by catalytic hydrogenation using tritium gas. The various labelled 3-ketosphingolipids will then be administered to developing rats intracerebrally. Various sphingolipids will be isolated from the brain, and the radioactivity will be measured. They will be hydrolyzed and the radioactivity in these components in the molecule of the products will be determined in order to find whether or not the sphingolipids are derived directly from these 3-keto derivatives without hydrolysis. In vitro studies of reduction of 3-ketosphingolipids to sphingolipids will be tested. The (C14) labelled 3-keto derivatives as well as 3-ketodihydrosphingosine will be used as the substrates and a preparation from young rat brain as the enzyme source. The assay procedures will include TLC separation of the sphingolipids and determination of their radioactivity. Alternatively, the enzyme reaction may be followed by change of concentration of reduced pyridine nucleotides. The enzymes will be purified by a variety of techniques including gel permeation and ion-exchange chromatography. The properties of the purified enzymes will be examined with special emphasis on substrate specificities and control mechanism. Bibliographic references: Murad, S. and Kishimoto, Y.: Effect of thyroid hormones on alpha-hydroxylation of long chain fatty acids in developing rat and mouse brain. Fed. Proc. 34: 346, 1975.